View source for SREBF1 Genotyping Protocol

==Primers==


*Srebf1 WT Fwd:  5' CCA TGT GCG CTC ACC CGA G 3'
*Srebf1 All Fwd:  5' CGA TCC GCT GTA GAG ACC CT 3'
*Srebf1 All REV:  5' CAT TCA GAG CAC CCG GTG AA 3'

==Primer Mixes==

Primer mixes are resuspended at 100uM by adding 10 times the nmol amount on the tube. (For example: nmol=32.4, add 324 uL of water to primer tube).

Add 2uL of each primer (WT Fwd, All Fwd, and All Rev) to 494uL of ddH2O to make a total volume of 500uL of diluted primer.

==PCR Reaction==

The PCR program is called "srebp" on the thermal cycler and is as follows:

*95°C 10 min 
* 95°C 10 s
* 57.5°C 30 s
* 72°C 45 s
**Go to step 2, 34 times (total of 35 cycles)
*72°C 5 min

==Bands==

For mutants 1 and 2 (m1 and m2) the WT bands are the same:  302 and 218
For m1: the mutant band is  at 277 (heterozygotes should have bands at 302, 218, and 277)
For m2: the mutant band is at 150 (heterozygotes should have bands at 302, 218, and 150)

Screen Shot 2016-08-30 at 12.48.27 PM



[[Category: Genotyping]]
[[Category: Mouse Work]]
[[ Category:PCR]]
[[ Category:DNA]]
[[ Category:Molecular Biology]]